Metal complexes of a multidentate cyclophosphazene with imidazole-containing side chains for hydrolyses of phosphoesters-bimolecular vs. intramolecular dinuclear pathway

Article abstract of DOI:10.1002/ejic.201000668

A multidentate imidazole-containing cyclophosphazene (Im₆Cpz) ligand was synthesized and its metal complexes (M_xIm₆Cpz; M = Zn^II, Cu^II, Co^II; x = 1, 2, 3) were prepared and used as phosphoe

Full text of DOI:10.1002/ejic.201000668

FULL PAPER
DOI: 10.1002/ejic.201000668
Metal Complexes of a Multidentate Cyclophosphazene with
Imidazole-Containing Side Chains for Hydrolyses of Phosphoesters –
Bimolecular vs. Intramolecular Dinuclear Pathway
Le Wang,[a,b] Yong Ye, Vasiliki Lykourinou, Alexander Angerhofer, Li-June Ming,*
[a]
[b]
[c]
[b]
and Yufen Zhao*[
a]
Keywords: Copper / Zinc / Hydrolysis / Homogeneous catalysis / Enzyme models
A
multidentate imidazole-containing cyclophosphazene
Cpz) ligand was synthesized and its metal complexes
than that for BNPP hydrolysis. An intramolecular dinuclear
pathway was revealed for the complexes Cu Im Cpz and
Cu Im Cpz; whereas an intermolecular dinuclear pathway
was observed for CuIm Cpz. Cu Im Cpz shows an order of
magnitude higher activity than the simple Cu complex of
the untethered ligand N-methylimidazole, Cu(MeIm) , sug-
gesting a possible contribution from the proximity effect in
the Im Cpz complexes. The high catalytic specificity of Cu
Im Cpz towards the phosphomonoester NPP suggests that it
(
(
Im
M
6
2
6
II
II
II
x
Im
6
Cpz; M = Zn , Cu , Co ; x = 1, 2, 3) were prepared
3
6
and used as phosphoesterase models towards hydrolysis of
the model substrates p-nitrophenylphosphate (NPP) and
bis(p-nitrophenyl)phosphate (BNPP) in 75% DMSO buffer
solution at pH = 7–11 at 37 °C. The hydrolysis of BNPP by
6
3
6
II
2
Cu
1.4ϫ10–
exhibits a tremendous selectivity toward NPP, showing
3
Im
6
Cpz exhibits enzyme-like saturation kinetics with kcat
6
3
-
5
–1
–1 –1
II
=
s
and kcat/K
m
= 0.0027
M
s . The Cu complex
6
can serve as a good model system and a blueprint for further
exploration of catalytic activity and specificity in phosphoes-
ter hydrolysis.
nearly a stoichiometric binding with kcat = 6.8ϫ10–
4 –1
s
under
saturation conditions and a significant second-order rate con-
stant kcat/K
–1
–1
4
d
= 136
M
s , which is 5.0ϫ10 times higher
Introduction
years for the autohydrolysis of a dibasic alkylphosphate at
[
5]
25 °C, its hydrolysis is among the most difficult reactions
Hydrolytic reactions promoted by mono-, di-, and multi-
nuclear metal complexes are of increasing importance in
biotechnology and medicine. Metal complex-mediated hy-
drolysis of phosphate esters provides valuable information
for modeling and elucidating the reactivity of metal-con-
taining nucleases as well as several dinuclear metallophos-
catalyzed by an enzyme. Therefore, the design and synthesis
of efficient and/or selective models of phosphoesterases and
nucleases are regarded as a great challenge due to their
value as biological tools and as chemotherapeutic agents.[
Various di- and multinuclear metal complexes have been
prepared and shown to exhibit potential cooperative effects
6]
[
1]
phatases involved in the cleavage of phosphoester bonds.
[
7]
between the metal centers. Cooperative effects have been
observed in multinuclear complexes of lanthanides (e.g.
Phosphate esters and their processes exist ubiquitously in
nature, such as nucleoside phosphates (nucleotides) as
building blocks of RNA and DNA, sugar nucleotides for
the glycosylation of oligosaccharides, and (de)phosphoryla-
tion in intracellular signaling and regulation.[ In the ab-
sence of a catalyst or enzyme, phosphoester hydrolysis is
extremely slow under normal conditions (e.g., the half life
IV
IV
III
Ce ), Sn , and Fe to efficiently hydrolyze the phospho-
[
8]
II
diester linkages in DNA and RNA. Dinuclear Cu com-
plexes have been reported to catalyze phosphate diester
cleavage with high catalytic activities (up to 1.6ϫ10 rate
2]
6
acceleration relative to the uncatalyzed reaction for the hy-
drolysis of model diester substrates at near physiological
7
[3]
of phosphodiester hydrolysis in DNA is 3ϫ10 years and
that of the activated phosphodiester bis(p-nitrophenyl)-
[
9]
conditions). Several other mono- and dinuclear complexes
also show that dinuclear complexes can be superior to mo-
[
4]
phosphate is about 2000 years at pH 7.0 and 25 °C ).
[
10]
nonuclear complexes for hydrolysis.
Moreover, with an estimated half life of an enormous 1011
Cyclotriphosphazene (Cpz) comprises alternating phos-
phorus and nitrogen atoms with a conjugate structure to
afford a planar ring. Its derivatives usually have biological
miscibility and degrade into small nontoxic molecules, thus
[
[
a] Department of Chemistry, Zhengzhou University,
Zhengzhou, 450052, China
E-mail: zicb@zzu.edu.cn
b] Department of Chemistry, University of South Florida,
[
11]
4
202 Fowler Avenue, CHE205, Tampa, Florida 33620-5250, are advantageous to biological research.
Moreover, the
USA
rich substitution chemistry at its phosphorus centers affords
the possibility for introducing other bioactive groups such
as metal-binding ligands for catalyst development.[ In
E-mail: ming@shell.cas.usf.edu
c] Department of Chemistry, University of Florida,
Gainesville, FL 32611, USA
[
12]
674
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2011, 674–682

Metal Complexes of a Multidentate Cyclophosphazene Ligand
II
view of current reports and as a continuation of our pre- as a function of [Cu ] (Figure 1, B, inset) can be fitted to a
[
13]
II
vious work,
we derivatized Cpz with six imidazole-con- Cu :Im Cpz 3:1 stoichiometry (solid trace) with a forma-
6
taining groups, (Im Cpz, Scheme 1) which mimic the well tion constant of 980 mm–1 for Cu binding to two imidazole
II
6
documented metal-binding side chain of histidine in metall- arms, consistent with the stoichiometry from the NMR ex-
oproteins. This derivative binds metal ions and mediates periment. The weak d-d transition around 720 nm indicates
II
metal-centered hydrolysis of phosphmonoester and phos- a type-2 Cu center.
phodiester with a much higher catalytic specificity towards
phosphomonoester. The mechanism has been revealed to
exhibit an intramolecular dinuclear pathway when
Cu:Im Cpz is 2:1 or 3:1. The high specificity towards phos-
6
phomonoester and the intramolecular dinuclear pathway
suggest that these complexes can serve as good model sys-
tems for metallophosphatases.
Figure 1. (A) Change of the chemical shift of the imidazole ring
protons of Im
8 at 7.772 ppm; Scheme 1) in [D
Im Cpz] = 5 mm. (B) Optical titration of Cu into 0.02 mm
Cpz in DMSO/HEPES 3:1 (100 mm buffer at pH 7.1) at 37 °C,
6
Cpz (᭢ = proton 6 at 6.928; ᭺ = 7 at 7.162; and ᭹
II
=
[
6
]DMSO due to Zn binding;
II
6
Im
6
monitored at 305 nm. The solid traces in (A) and (B) are the best
fits to the metal:ligand 3:1 stoichiometry.
The nature of the CuII center in the complex is revealed
with electron paramagnetic resonance (EPR) spectroscopy
Scheme 1.
(
Figure 2), in which the unpaired electron in the d
x2–y2
or-
II
bital of Cu affords an EPR spectrum with g Ͼg (or gz
ʈ
Ќ
Ͼg and g ). The EPR spectral features of the complexes
x
y
Results and Discussion
with Cu:Im Cpz of 1:1, 2:1, and 3:1 are similar with g
6
ʈ
Metal binding of Im Cpz
≈ 2.27–2.30 (A ≈ 540–570 MHz) and g ≈ 2.07–2.10 (AЌ
6
ʈ
Ќ
Due to the existence of multiple metal binding sites in ≈ 30–40 MHz), suggesting a similar coordination environ-
Im Cpz that can potentially form metal complexes with ment of the three metal centers with a nitrogen-rich coordi-
6
[
14]
multinuclear centers, the complexes may provide mechan- nation sphere.
istic information about metal-mediated reactions, such as broaden with increasing Cu binding, clearly shown in Cu3
However, the gʈ features significantly
II
-
intermolecular vs. intramolecular and mononuclear vs. di- Im Cpz, suggesting the presence of a magnetic interaction
6
nuclear catalytic pathways. There are two possibilities for possibly attributable to the proximity of the paramagnetic
II
[15]
metal binding to Im Cpz, “facial binding” with each metal Cu centers.
The proximity of the metal centers would
6
bound to three imidazole-containing arms on each side of favor dinuclear catalysis. For spectral comparison, the all
II
the Cpz ring or “extended binding” with each metal bound N(His) coordination sphere in Cu -substituted carbonic
to two arms from the same phosphorus atom. The 1
NMR spectra of the ligand in the presence of different and g = 2.31 and g = 2.06 and Cu,Zn-superoxide dis-
H
anhydrase B shows an electronic transition band at 750 nm
[
16]
ʈ
Ќ
II
amounts of Zn in [D ]DMSO were obtained. The flux- mutase exhibits a 680 nm band and g = 2.268 and g
=
6
II
ʈ
Ќ
[17]
ional nature of the Zn Im Cpz complex is attributed to 2.087.
chemical exchange between the Zn bound and free forms
leading to gradual shift of the H NMR signals of Im Cpz
during the Zn titration. The change in the chemical shifts sis can be determined from Cu titration and Job plot
is plotted as a function of [Zn ], which is fitted to the bind- analysis
ing of 3 equiv. of Zn to Im Cpz with an apparent forma- BNPP. Detailed kinetic analyses and the mechanisms are
tion constant of 1100Ϯ730 m for Zn binding to two discussed later. The activity Job plots
6
II
Metal Binding Stoichiometry of Im Cpz for Catalysis
6
1
II
The Cu binding stoichiometry of Im Cpz during cataly-
6
6
II
II
II
[18]
by monitoring the activity towards NPP and
II
6
–
1
II
[19]
II
with a maximum
imidazole arms (Figure 1, A). Moreover, the addition of at XCu ≈ 0.75 are best fitted to Cu :Im Cpz 3:1 (i.e.,
6
II
Cu to Im Cpz in DMSO/4-(2-hydroxyethyl)-1-piperazine- XCu:XIm6Cpz 0.75:0.25; Figure 3), indicating that Cu3-
6
ethanesulfonic acid (HEPES) (3:1) at pH 7.1 results in a Im Cpz is the predominant active species towards hydroly-
6
gradual increase in absorption in the region of 250–350 nm sis of NPP and BNPP and confirming that extended rather
(Figure 1, B). A plot of the change in absorption at 305 nm than facial binding is the preferred metal binding mode.
Eur. J. Inorg. Chem. 2011, 674–682
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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675

L.-J. Ming, Y. Zhao et al.
FULL PAPER
7
5% DMSO/buffer solution is equivalent to that at pH 7.5
1.5
in buffer solution, representing a further 10 -fold enhance-
ment to reach 6.3ϫ10 -fold, in aqueous solution.
4
Figure 2. X-band EPR spectra of (solid traces from top to bottom
at 260 mT) Cu(MeIm)
spectra (dashed traces) for Cu(MeIm)
2
and Cu
x
Im
6
Cpz (x = 1, 2, 3), and simulated
(g = 2.07, g = 2.318, A
Cpz (g = 2.069, g = 2.103,
z
573 MHz).
2
Ќ
ʈ
Ќ
=
18 MHz, A
= 2.268, A
ʈ
= 490 MHz) and CuIm
= A = 40 MHz, A
6
x
y
g
z
x
y
Figure 4. Plot of the initial hydrolytic rate of BNPP by 0.1 mm Cu
Im Cpz fitted to Equation 2 (solid trace). The dashed traces is the
fit to Hill’s equation. Inset is shown the initial rate for the hydroly-
sis of 3 mm BNPP by different amounts of Cu Im Cpz to afford
obs. Conditions: 75% DMSO in 100 mm CHES at pH 9.0 and
7 °C.
3
-
6
3
6
k
3
Figure 3. Activity Job plot for the determination of the stoichiome-
II
try of the Cu complex of Im
6
Cpz. The total concentration of
Cpz] + [Cu ] is 0.4 mm for NPP (᭺, left scale) and BNPP (᭹,
right scale) hydrolysis, respectively, in 75% DMSO solution of
II
[Im
6
1
9
00 mm N-cyclohexyl-2-aminoethanesulfonic acid (CHES) at pH
Figure 5. The pH dependence of the initial rate for the hydrolysis
of 3.0 mm BNPP by Cu Im Cpz at 37 °C (᭹ and solid fit trace)
and of the molar absorptivity of p-nitrophenol (᭡ and dashed fit
trace). The molar absorptivities at different pH values were used
for the determination of the reaction rates at different pH.
.0 and 37 °C. The solid traces are the best fits to the stoichiometry
II
of Cu :ImCpz 3:1; while the dashed traces represent fits to 1:1
stoichiometry.
3
6
Phosphoester Hydrolyses by Metal Complexes of Im Cpz
The rate of BNPP hydrolysis by Cu Im Cpz with respect
6
3
6
The rate of hydrolysis of 3 mM BNPP increases with to [BNPP] is not linear and reaches a plateau at high
addition of Cu Im Cpz (inset, Figure 4), showing a pseudo- [BNPP] (Figure 4), indicative of pre-equilibrium kinetics
3
6
–
6 –1
first order rate constant of kobs = 4.5ϫ10 s . Although analogous to enzyme catalysis [Equation (1)] with the rate
BNPP is considered to be quite accessible to hydrolysis due law expressed as Equation (2). The data for BNPP hydroly-
to the good leaving group p-nitrophenol, its hydrolytic rate sis can be fitted well to Equation (2) to yield the kinetic
–
11 –1
–5 –1
is still extremely low with k = 1.1ϫ10
s
at pH 7.0 and constants kcat = 1.4ϫ10
s
(as the first order rate con-
o
[
4]
–9 –1
2
9
1
5 °C, which can be estimated to be 2.2ϫ10 s
.0 and 37 °C considering OH as the nucleophile (i.e., a rate constant (or the catalytic efficiency) kcat/Km
0-fold change in rate per pH unit and about two-fold rate 0.0027 m s at low [S] ϽϽ K (Figure 4 and Table 1). Due
at pH stant at [S] ϾϾ K ) and K = 5.2 mm, and a second order
m
m
–
=
–1 –1
m
change per 10 °C). Thus, a significant pseudo-first order to the change in the apparent pK by 1.5 units under the
a
3
rate enhancement kobs/k = 2.0ϫ10 can be obtained with experimental conditions relative to that in an aqueous solu-
o
respect to the noncatalyzed hydrolytic reaction at pH 9.0. tion (discussed below), the rate constants are comparable
However, since the reactions were conducted in 75% to those obtained in aqueous solutions at pH 7.5 and the
DMSO/buffer solution, the apparent ionization constant as rate enhancements are thus 30-fold (i.e., 101 ) higher than
.5
determined by the pK of p-nitrophenol increases by 1.5 the values obtained from aqueous solutions at pH 9.0. A
a
[
20]
5
units (cf. Figure 5). Thus, the rate constant at pH 9.0 in the catalytic proficiency of 2.0ϫ10 is thus obtained for the
676
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Metal Complexes of a Multidentate Cyclophosphazene Ligand
hydrolysis of BNPP in terms of first order rate constants ing and increase in hydrolytic rate of phosphoesters. As
1
1
(
kcat/k ) and 1.1ϫ10 in terms of the second order cata- phosphoester substrates bind to the metal center, DMSO
o
[
21]
lytic efficiency
(kcat/K )/k at pH 9.0 in 75% DMSO influence on catalysis other than the pK is expected to be
m w a
buffer solution, wherein kw = k /13.9 with 13.9 m being small.
o
[
H O] in 75% aqueous DMSO solution. The kinetics for
Zn Im Cpz is also active towards BNPP hydrolysis (k
2
3 6 cat
the hydrolysis of BNPP is better fitted to Hill’s equation = 1.8ϫ10–6 s ; Table 1), yet nearly an order lower than
–1
II
with a small Hill’s coefficient θ = 1.5 (dashed trace, Fig- Cu Im Cpz. Despite its high Lewis acidity, Cu does not
3
6
ure 4), suggesting the possible presence of cooperativity activate many Zn enzymes such as carboxypeptidase A and
[
25]
which may be attributable to dinuclear catalysis discussed carbonic anhydrase.
However, a few Zn hydrolases can
II
[26]
[27]
later.
be significantly activated by Cu (BP10,
and astacin ). Presumably the more restricted tetragonally
distorted Cu coordination relative to the flexible Zn co-
ordination may account for the low activities in Cu -substi-
tuted Zn enzymes. In simple complexes, the coordination is
more flexible relative to that in metalloenzymes which may
cause Cu to exhibit high hydrolytic activities due to its
higher Lewis acidity than Zn .
substituted Zn hydrolases are catalytically comparable to
serralysin,
[
28]
II
Table 1. Hydrolytic activities of M
x
Im
6
Cpz toward phosphoes-
II
[
a]
ters.
II
Complex
k
cat
K
[mm]
m
k
cat/K
m
–
1
–1 –1
[s ]
[M s ]
[
b]
–5
–6
–3
II
Cu
Zn
Co
Cu(MeIm)
3
Im
Im
Im
6
Cpz
Cpz
(1.4Ϯ0.2)ϫ10
(1.8Ϯ0.5)ϫ10
5.2Ϯ1.0
1.1Ϯ0.7
3.6Ϯ0.7
9.3Ϯ3.1
0.005Ϯ0.001
0.005Ϯ0.002
0.17Ϯ0.05
0.32Ϯ0.05
0.76Ϯ0.16
2.7ϫ10
[b]
–3
3
6
1.6ϫ10
II [29]
II
Conversely, most Co -
[
b]
–7
–4
3
6
Cpz
(6.8Ϯ0.6)ϫ10
1.9ϫ10
[b]
–6
–4
2
(2.1Ϯ0.5)ϫ10
2.3ϫ10
[
c]
–4
[d]
[e]
Cu
Cu
CuIm
Zn Im
Cu(MeIm)
3
Im
Im
Cpz
6
Cpz
(6.8Ϯ0.2)ϫ10
136
82
the native enzymes, attributable to their similar coordina-
However, Co Im Cpz shows a low ac-
tivity towards BNPP hydrolysis with kcat = 6.8ϫ10
[
c]
–4
[d]
[e]
[25]
2
6
Cpz
(4.1Ϯ0.2)ϫ10
tion geometries.
3 6
[
c]
–4
[f]
(2.64Ϯ0.16)ϫ10
1.6
0.54
–7 –1
6
s
[c]
–4
3
6
Cpz
(1.73Ϯ0.07)ϫ10
[
c]
–5
–2
and K = 3.6 mm (Table 1). The smaller kcat value is proba-
m
2
(6.1Ϯ0.4)ϫ10
8.1ϫ10
2+
bly due to the relatively low Lewis acidity of the Co ion
[
a] Conditions: reaction volume = 1.0 mL in 100 mM CHES (75%
DMSO) at pH = 9.0 and 37 °C, [M Im Cpz] = 0.1 mm, [BNPP] =
.25–6.0 mm, [NPP] = 0.01–6.0 mm. [b] BNPP as the substrate. [c]
obtained from cycles due to the inertness of the Co3 center.
NPP as the substrate. [d] Dissociation constant K
the equilibrium: complex–NPP h complex + NPP. [e] Second-order
rate constant obtained from kcat/K . [f] The bimolecular dinuclear
center is considered as a single unit in the fit to yield K
.11Ϯ0.03 mm.
III
as opposed to Co complexes with higher Lewis acidities
x
6
that exhibit high k values which, however, lacks turnover
0
cat
+
[9,30]
d
The kcat and kcat/Km values of the complexes herein
towards BNPP hydrolysis (Table 1) are higher than or com-
parable to several mono- and multinuclear metal complexes
and metallopolymers. For example, [(1,4,7-triazacyclo-
d
d
=
0
+
–6 –1
Since metal-coordinated water molecules play a key role nonane)CuOH] exhibits k
= 9ϫ10
s
at 50 °C and
obs
–1 –1
in metal-mediated hydrolyses, the effect of pH on BNPP pH 7.2 and k = 8ϫ10– m s at 35 °C;
4
[31]
Cu(bipyr-
2
2+
–5 –1
at pH 6.5 and 75 °C;[32]
hydrolysis by Cu Im Cpz was investigated. A typical sig- idine) , k
= 4.2ϫ10 s
3
6
obs
moidal rate–pH profile was obtained and fitted to a single Cu {N,N’,N,N’-bis-[(2-hydroxybenzyl)(2-pyridylmethyl)]-2-
2
proton ionization process to give pK = 8.73 by considering ol-1,3-propanediamine}(μ-OAc)(H O) Cl ,
kcat
=
a
2
2
2
the deprotonated form as the active species (Figure 5), pre- 5.4ϫ10–
5
s
–1
and kcat/Km = 0.027 m s at pH 7.0 and
–1 –1
–
[33]
sumably due to a metal-bound nucleophilic OH . Since 40 °C;
[Cu {p-6,6’-di-[bis-(2-pyridylmethyl)amino-2-
2
II
II
2+
–6 –1
both Cu and Zn bind well to the ligand at pH 7.1 as methylpyridyl]benzene}(OH) ] , k
= 1.14ϫ10
s
in
2
obs
34]
[
shown with NMR and optical methods, the increase in rate 30% DMSO at pH 8.4 and 55 °C;
dinuclear [Zn {2,7-
2
in the rate–pH profile cannot be due to ligand ionization bis[2-(2-pyridylethyl)aminomethyl]-1,8-naphthyridine}(μ-
2
+
–7 –1
and metal binding but must be due to the deprotonation of OH)(μ-O Ph )] , k
= 6.5ϫ10
s
(20% acetonitrile,
2
2
obs
35]
[
II
the nucleophilic water. The relatively high pK value com- pH 7.0, and 40 °C);
reflects cyclotetraamine ligands afford (1.1–3.1)ϫ10 m s under
a low Lewis acidity of the metal center in Cu Im Cpz, various conditions;
a few trinuclear Zn complexes of
a
pared to those of many other metal complexes[
29,22]
–4 –1 –1
[
36]
III
and an acridine-containing di-Fe
3
6
2
+
–5 –1
which is inconsistent with the nature of the Cu ion. Since complex exhibits k
= 5.2ϫ10
s
and kcat/Km
=
cat
–1
–1
[37]
the pK is significantly affected by the environment of the 0.017 m s in 10% DMSO at pH 7.36 and 50 °C. A chi-
a
II
–5 –1
ionizable group, as found in the diverse environments in tosan-based Cu complex exhibits k = 8.33ϫ10
s
and
cat
[
23]
–3 –1 –1
II
[38]
proteins, the influence of DMSO on pK was addressed. kcat/Km = 1.17ϫ10 m s (pH 11 and 30 °C), two nu-
The pK of the hydrolytic product shifts to a higher value cleobase-containing Cu
a
polymers show
k
=
and
a
cat
–1 –1
–6
–1
–1
3.04ϫ10–5 m s
by 1.5 units from 7.15 to 8.64 in 75% DMSO/buffer solu- 1.13ϫ10
s
s
and kcat/Km
=
tions. Accordingly, the pK value of the nucleophilic water 2.77ϫ10–7
and 4.5ϫ10 m s (50% methanol, pH
and the Cu complex of a pyrazolyl-Cpz-
–5 –1 –1
a
[
39]
II
would be ca. 7.2 in buffer solutions. A vibrational spectro- 8.0, 30 °C),
scopic study indicates that DMSO elongates the ester bond, containing polymer (without a flexible linker attached to
presumably by decreasing solvation on the terminal oxygen the Cpz ring) exhibits k = 2.93ϫ10–6
s
–1
and k /Km
=
cat
cat
[40]
[
24]
–3 –1 –1
atoms;
however, the change in the ester bond energy is 3.96ϫ10 m s (50% methanol, pH 8.0, 30 °C), and a
III
–6 –1
small compared to the changes in energy associated with metallopolymer Fe -P1, k = 2.2ϫ10
s
and kcat/Km
For comparison,
trostatic interactions are proposed to cause bond lengthen- the catalytic specificities kcat/Km of the alternative activity
=
cat
the different reactivities of various esters. Nevertheless, elec- 4.9ϫ10–3 m s at pH 8.0 and 25 °C.
–1
–1
[19b]
Eur. J. Inorg. Chem. 2011, 674–682
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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677

L.-J. Ming, Y. Zhao et al.
FULL PAPER
–
1
–1
9.0ϫ10–7 s )
–1 [46]
and a ZnII complex of a histidine mimic
of a few enzymes toward BNPP hydrolysis are 0.05 m s
for Escherichia coli alkaline phosphatase,[ 11.4 m s for shows a slight preference towards NPP hydrolysis than
41]
–1 –1
[
42]
–5
Burkholderia phosphonate monoester hydrolase,
and BNPP hydrolysis at pH 7.0 and 50 °C (k = 2.1ϫ10 vs.
obs
II
–
1
–1
–5 –1 [47]
0
.5–100 m s for Streptomyces aminopeptidase and its 1.1ϫ10 s ); whereas a dinuclear Cu complex exhibits
metal derivatives; whereas a native phosphodiesterase en- a catalytic specificity toward NPP (k = 1.45ϫ10
[
43]
–3 –1
s
and
cat
7 –1 [48]
–
coded by ElaC shows
a
much higher value of K = 1.0 mm) over BNPP (2.8ϫ10 s ) and a dinuclear
m
II
–
1
–1 [44]
1480 m s .
Cu complex of a dicyclic amine ligand shows a signifi-
The hydrolysis of the phosphomonoester substrate NPP cantly higher catalytic specificity towards NPP than BNPP
by Zn Im Cpz follows saturation kinetics (see ♦ in Figure 6) (2.2ϫ10– vs. 9.4ϫ10 m s ).
2
–4 –1 –1 [49]
However, the mononu-
and kcat/Km = 0.54 m s , clear Cu complex of a dicyclic amine ligand does not show
3
6
–
4
–1
–1 –1
II
to afford kcat = 1.73ϫ10
s
which are 94 and 340 times, respectively, higher than those such selectivity and a mononuclear Cu–cycloamine com-
for BNPP hydrolysis (Table 1), suggesting its catalytic speci- plex exhibits 500 times less activity toward NPP than
[
31]
ficity toward phosphomonoesters. NPP hydrolysis by Cu3- BNPP,
suggesting the importance of a di- or multinu-
Im Cpz also reaches a plateau at high [NPP] (see ᭹ in Fig- clear center for hydrolysis of phosphomonoesters as ob-
6
[
42]
ure 6), suggesting a pre-equilibrium mechanism as in Equa- served in the enzyme alkaline phosphatase.
tion (1). However, the data cannot be fitted well with Equa- The rate constant kcat for NPP hydrolysis by Cu Im Cpz
3
6
tion (2) (solid traces, Figure 6). The kinetic profile shows was determined at different temperatures (Figure 7), from
nearly stoichiometric NPP binding to Cu Im Cpz, indicat- which the activation energy E = 49.6 kJ/mol was deter-
3
6
a
ing a very high catalytic specificity. As such, the rate law of mined with the Arrhenius equation which yields the en-
‡
–1
Equation (2) is no longer valid for the mechanism in Equa- thalpy of activation ΔH = 50.5 kJmol . The Gibbs free
‡
–1
tion (1), wherein the catalyst-bound substrate (CS) is as- energy of activation ΔG = 94.3 kJmol was obtained from
sumed to have a lower concentration than the free form, kcat according to the Eyring–Polanyi equation and the en-
‡
–1 –1
i.e., [S] ϾϾ [CS] to afford the term [S] ([E] – [CS]). Thus, tropy of activation ΔS calculated to be –144.8 Jmol K .
o
o
o
the steady state treatment of Equation (1) in the form of a The thermodynamic parameters for the hydrolysis of NPP
quadratic equation k ([S]o – [CS])([E]o – [CS]) = (kcat by Cu Im Cpz and CuIm Cpz were also determined (E =
+
1
2
6
6
a
–1
‡
–1
‡
k )[CS] is necessary (dashed traces; Figure 5), and affords 68.7 and 77.3 kJmol , ΔH = 65.5 and 74.5 kJmol , ΔG
–
1
–
4
–1
–1
‡
kcat
=
6.8ϫ10
s
under saturation conditions of = 94.9 and 96.0 kJmol , and ΔS = –98.6 and –72.2
–
1
–1
[
S] Ͼ [Cu Im Cpz], a very small dissociation constant K
Jmol K , respectively). Since the NPP hydrolysis by Cu -
o
3
6
d
x
=
5 μm for the CS complex [NPP(Cu Im Cpz)], and a sig- Im Cpz follows a pre-equilibrium kinetic pattern to form
3 6 6
–1
–1
‡
nificant catalytic specificity of kcat/Kd
=
136 m s
Table 1). The kcat value is 4.1ϫ10 times larger than NPP that the transition state CS is much better organized than
autohydrolysis in 0.2 m Tris buffer at pH 9.3 (k the CS complex. The hydrolysis of bound NPP in the inert
This kcat value is ca. 50 times higher, complex cis-[Co(en) (OH)(NPP)], which mimics the Mi-
the Michaelis CS complex, the negative ΔS value reflects
4
‡
(
=
o
–
8 –1 [45]
1.67ϫ10 s ).
2
4
‡
–1 –1 [50]
whereas the catalytic specificity kcat/Km is 5.0ϫ10 times chaelis CS complex, has ΔS = –65 Jmol K , close to
higher, than those for BNPP hydrolysis by Cu Im Cpz that for NPP hydrolysis by CuIm Cpz. Conversely, NPP au-
3
6
6
‡
–1 –1
(Table 1).
tohydrolysis showed ΔS = –18.8 Jmol K and was sug-
gested to follow a dissociative mechanism
[
51]
which never-
[
52]
theless was questioned.
Crystallographic “snapshots” of
phosphoserine phosphatase with a bound substrate (D11N
mutant), transition state analogues, and the phosphate
[
53]
product
revealed that the transition state analogues in
Figure 6. Plot of the initial hydrolytic rate of NPP by 0.1 mm CuII
complexes (left and bottom scales) Cu ImCpz (᭹), Cu ImCpz (᭺),
and CuImCpz (᭢) and Zn ImCpz (♦, top and right scales). The
3
2
3
solid traces are fits to Equation (2) while the dashed traces are fits
to a tight-binding mode. Conditions: 75% DMSO in 100 mm
CHES at pH 9.0 and 37 °C.
Figure 7. Temperature dependence of kcat for the hydrolysis of NPP
by Cu Im Cpz (᭹), Cu Im Cpz (᭺), and CuIm Cpz (᭢) in 75%
DMSO of 100 mm CHES at pH 9.0. The activation energy E is
a
A dinuclear ZnII complex of triazacyclononane linked
with a spacer shows a slightly higher hydrolytic activity to-
3
6
2
6
6
–
6 –1
ward NPP than BNPP (kobs
=
2.2ϫ10
s
vs. obtained by directly fitting to the Arrhenius equation.
678
www.eurjic.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2011, 674–682

Metal Complexes of a Multidentate Cyclophosphazene Ligand
the enzyme showed the reaction coordinate distances of was modeled with geometry-optimized molecular mechan-
3
.59 and 3.56 Å which are significantly shorter than the dis- ics calculations, which shows that two metal centers can be
–
2–
tance of 4.9 Å estimated for the fully dissociative mecha- in close proximity with a possible OH /O bridging ligand
nism, suggesting an associative mechanism.
and a pseudo-twofold symmetry of the two arms (Figure 9).
Such an arrangement suggests a probable intramolecular
Mechanistic Investigation
Because Im Cpz is capable of binding up to three equiva- dinuclear catalytic pathway. Although such a dinuclear cen-
6
lents of metal, it is therefore important to gain further in- ter during catalysis does not need to be present in the rest-
sight into possible interactions among the three metal cen- ing state of the complexes, the gradual increase in broadness
II
ters in substrate binding and hydrolytic activity. The kinetic of the EPR spectra with increasing amount of Cu in the
traces for hydrolysis of BNPP and NPP can be better fitted complex suggests the presence of magnetic interactions due
II
by the use of Hill’s equation with Hill’s coefficients θ = 1.47 to the proximity of the Cu centers.
and 1.42, respectively, indicative of cooperativity and a di-
–
nuclear pathway. The charged ϾPO2 moiety in BNPP and
NPP is feasible for interaction with two metal centers to
render a dinuclear pathway.
The (MIm Cpz):S ratio in the pre-equilibrium MIm Cpz
6
6
+
S h (MIm Cpz)–S can be determined by means of the
6
[19]
mechanistic Job plot
using hydrolytic activity (which is
proportional to [(MIm Cpz)–S]; Equation (1)) instead of
6
optical density as the output. The maximum in the mechan-
istic Job plot thus reflects the (MIm Cpz):S ratio in the ca-
6
3 6
talysis. The data show a maximum at Xcomplex ca. 0.5 for Figure 9. A plausible structure of the Cu Im Cpz complex with an
intramolecular dinuclear center obtained from molecular mechan-
ics calculations (BioMedCAChe 6.1, Fujitsu). One coordinated
NPP hydrolysis by Cu Im Cpz and Cu Im Cpz which can
2
6
3
6
be best fitted with (Cu Im Cpz):NPP = 1:1 (Figure 8), i.e.,
x
6
II
–
water on each Cu center is assigned as the deprotonated OH and
Xcomplex:XNPP = 0.5:0.5. In the case of Cu Im Cpz, the 1:1
–
2
6
the dinuclear site is bridged by an oxide. A Cl ion was added as a
ratio may suggest an intermediate (Cu Im Cpz)–NPP with counterion to balance the charge.
2
6
an internal dinuclear center formed by two Cu bound arms
or (Cu Im Cpz) –NPP with two dinuclear centers formed
The kinetic pattern of NPP hydrolysis by CuIm Cpz is
2
6
2
2
6
by two metal complexes (which is thermodynamically unfa- significantly different from those by Cu Im Cpz and Cu -
2
6
3
vorable). In the case of Cu Im Cpz, the substrates may bind Im Cpz (Figure 6), suggesting different catalytic mecha-
3
6
6
to an intramolecular dinuclear metal center as in (Cu2- nisms. For example, CuIm Cpz would not be likely to form
6
Im Cpz)–NPP, which would leave one mononuclear arm. an intramolecular dinuclear center as Cu Im Cpz and Cu -
6
2
6
3
The higher activity of Cu Im Cpz than Cu Im Cpz Im Cpz do. The mechanistic Job plot for NPP hydrolysis
3
6
2
6
6
(
Table 1) may be due to a higher probability of collision by CuIm Cpz shows a maximum at X
ca. 0.65, which
6
complex
attributable to the three metal centers, or because all the can be fitted with a CuIm Cpz:NPP ratio of 2:1 (᭹ and
6
three metal centers are active.
dashed trace; Figure 8), i.e., Xcomplex/XNPP ca. 0.65:0.35,
suggesting a dinuclear center formed by two complex mole-
cules. Accordingly, Cu Im Cpz can form an intramolecular
3
6
dinuclear center as in Cu Im Cpz and an intermolecular
2
6
dinuclear center as in CuIm Cpz for two parallel pathways
6
during hydrolytic catalysis of NPP. In such a case, the Cu3-
Im Cpz:NPP ratio would be 2:3, which is thermodynami-
6
cally less favorable. The active intermediate during Cu3-
Im Cpz catalysis is likely to be NPP–(Cu Im Cpz)–NPP–
6
3
6
(
Cu Im Cpz) or to simply follow both intra- and intermo-
3 6
lecular dinuclear pathways. Indeed, the k value of Cu3-
cat
–4 –1
Im Cpz (6.8ϫ10 s ) is the sum of those of Cu Im Cpz
6
2
6
–1
(
4.1ϫ10–4 s ) following an intramolecular pathway and
–4 –1
CuIm Cpz (2.64ϫ10 s ) following an intermolecular
6
pathway.
Figure 8. Mechanistic Job plot of Cu
and CuIm Cpz (᭹) towards the hydrolysis of NPP with a constant
total concentration ([complex] + [NPP]) of 0.1 mm (25 °C, pH =
.0 75% DMSO). The data are fitted to the stoichiometry of com-
plex:NPP = 1:1 (solid traces) or 2:1 (dashed traces).
3 6 2 6
Im Cpz (᭢), Cu Im Cpz (᭺),
Cu(MeIm)2
6
The mechanism of the Cu Im Cpz complexes are further
x
6
II
investigated with a simple mimetic system, the Cu complex
with MeIm which represents a metal center not tethered to
the Cpz core. The binding of MeIm to Cu is demonstrated
9
II
Owing to the very low solubility of the metal complexes, by an electronic transition at 700 nm which reaches a pla-
attempts to grow crystals were unsuccessful. Possible ar- teau at the addition of 2 equiv. of MeIm, indicative of the
rangement of the six arms of Im Cpz upon metal binding formation of the complex Cu(MeIm)2 in solution (Fig-
6
Eur. J. Inorg. Chem. 2011, 674–682
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
679

L.-J. Ming, Y. Zhao et al.
FULL PAPER
ure 10, A). Cu(MeIm) exhibits an EPR spectrum with g = ions. The resulting complexes can effectively catalyze the
2
ʈ
2
.318, A = 490 MHz, g = 2.074, and AЌ = 18 MHz (Fig- hydrolysis of the phosphomonoester NPP and phosphodi-
ʈ
Ќ
ure 2). Addition of more MeIm results in a slight shift of ester BNPP in 75% DMSO in buffer, with a tremendous
4
the electronic transition to 670 nm (inset, Figure 10, A), selectivity towards NPP over BNPP by 5.0ϫ10 times in
suggesting the formation of a different complex. For com- terms of catalytic specificity kcat/Km for Cu Im Cpz. Com-
3
6
II
parison, the hydrolytic activities of Cu(MeIm)2 towards pared with an untethered imidazole complex of Cu , Cu -
2
BNPP and NPP were determined under the same condi- Im Cpz and Cu Im Cpz exhibit up to two-order higher
6
3
6
tions as for Cu Im Cpz (Figure 10, B), showing k values rates toward phosphoester hydrolysis in terms of kcat, sug-
3
6
cat
up to two orders of magnitude smaller than Cu Im Cpz gesting that the proximity of metal centers in these com-
3
6
and Km values about an order higher (Table 1). As Km
=
plexes are catalytically significant analogous to the multinu-
(
kcat + k )/k , a large decrease in k and a significant in- clear center in alkaline phosphatase. The catalysis follows
–1
1
cat
crease in Km reflect a large increase in the dissociation of an intramolecular pathway with the substrate NPP bound
II
the catalyst–substrate complex (or poor substrate binding). to two Cu centers in Cu Im Cpz and Cu Im Cpz, but an
2
6
3
6
This observation demonstrates that Cu Im Cpz is superior intermolecular pathway in CuIm Cpz with the substrate
3
6
6
to the simple complex Cu(MeIm) in terms of substrate re- bound to two complexes in which the intramolecular path-
2
cognition and hydrolytic catalysis. The stoichiometry of the way is not available. These complexes display significant
[
Cu(MeIm) ]–NPP intermediate was also determined with catalytic proficiencies and unique catalytic mechanism and
2
the mechanistic Job plot. The data were better fitted to the can also serve as new structural motifs for further explora-
stoichiometry of Cu(MeIm) :NPP = 2:1 (Figure 10, C), rep- tion of chemical modeling.
2
resenting a intermolecular dinuclear pathway as found in
the case of CuIm Cpz catalysis.
6
Experimental Section
6
Materials: Im Cpz was synthesized following a previously estab-
lished method.[ All reagents are commercially available, solvents
distilled and dried with standard procedures, and aqueous solu-
tions prepared from 18 MΩ deionized or distilled water. The 75%
DMSO buffer solutions were prepared by direct mixing DMSO
with the buffer at a desired pH, HEPES (pH 7.0–8.0) and CHES
13]
(
a
pH 9–10.0) at 100 mm. The pK value (7.15) of the hydrolytic prod-
uct p-nitrophenol changes with the addition of DMSO and be-
comes 8.64 in the presence of 75% DMSO, which affect its molar
absorptivity at different pHs and are calibrated based on the fully
deprotonated form.
Spectroscopic and Kinetic Measurements: NMR spectra were re-
corded with a Varian INOVA500 spectrometer. Electronic spectra
and all kinetic measurements were performed with a Varian Cary
50 spectrophotometer. EPR spectra were obtained with a Bruker
Elexsys E580 X-band spectrometer at ca. 5–6 K using an Oxford
ESR900 cryostat and the Bruker standard rectangular TE102 reso-
nator, typically with a microwave frequency ca. 9.4 GHz, field mod-
ulation around 2 G, and time constant/conversion time settings of
4
0/80 ms. The g and A values were obtained from spectral simula-
[54]
tions performed with the “easyspin” toolbox for Matlab.
The initial rates of the hydrolysis of all the phosphoester substrates
were monitored spectrophotometrically between 10 and 60 min
through the change at 419 nm due to the production of p-nitro-
phenolate (measured in 75% DMSO/buffer of 100 mm at 37 °C, ε
= 15,600 m cm at pH 9.0). Various amounts of the complex and
substrates were used in the reactions separately, from which the rate
laws were determined and the rate constants obtained by means of
nonlinear regression. The kinetics can be described as the binding
of the substrate S to the metal center to form an intermediate S–
Figure 10. (A) Optical titration of MeIm into 4 mM CuII in meth-
anol at 37 °C, monitored at 700 nm. The solid trace is the fitting
to the formation of a 1:2 (ML
᭡; right scale) and NPP (᭹; left scale) by Cu(MeIm)
anistic job plot of NPP hydrolysis by Cu(MeIm) with [complex] +
NPP] = 1.0 mm. The data are fitted to Cu(MeIm) :NPP 2:1 (solid
2
) complex. (B) Hydrolysis of BNPP
–
1
–1
(
2
. (C) A mech-
2
[
2
trace) and 1:1 (dashed trace). Conditions: 75% DMSO in 100 mm
CHES at pH 9.0 and 37 °C.
[
Cu
Equation (1). The pre-equilibrium rate law is expressed as Equation
2) with the assumption that the amount of bound S is much less
than that of free S in solution, in which K = (k–1 + kcat)/k is
3 6
Im Cpz] complex, followed by conversion to the products; see
(
Conclusion
m
1
A Cpz core functionalized with six imidazole-containing the virtual dissociation constant of the bound S, equivalent to the
arms has been shown to bind up to three divalent metal Michaelis constant in enzyme catalysis.
680
www.eurjic.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2011, 674–682

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